Lignocellulosic materials, such as agro-industrial residues (sugarcane bagasse) or forestry are an important source of renewable biomass for the biofuel industry; Lignocellulose comprises cellulose, hemicellulose, and lignin, where lignin functions as a cementing agent between cellulose and hemicellulose fibers, protecting them from microbial and enzymatic attack. Lignin is a complex, aromatic biopolymer consisting of phenolic units mainly p-hydroxyphenyl, guaiacyl, and syringyl-type phenylpropane, which are linked together by ether and carbon-carbon bonds. Sources of lignocellulose biomass include SugarCane Bagasse (SCB); barley straw and wheat straw.
SCB is a fibrous residue of cane stalks that is left over after the crushing and extraction of the sugar rich juice from sugarcane (Saccharum officinarium). SCB mainly consist of cellulase 51% (w/w), hemicellulose 23% (w/w), and insoluble lignin 22% (w/w), and may be used as a source of cellulosic ethanol production.
The utilization of lignocellulose, such as SCB, barley and wheat straw, for biofuel production is hampered by difficulties faced in degrading these recalcitrant substrates, where the presence of lignin in the lignocellulose materials is suggested to retard the cellulases during their enzymatic hydrolysis of cellulose. Pre-treatment of the lignocellulose biomass is used to partially solubilize the biomass and thereby enhance the accessibility of the cellulose to enzymatic attack. Steam Explosion (STEX) uses a combination of high-pressure steam, followed by an explosive decompression, to partially hydrolyze hemicelluloses, which also serves to temporarily solubilize and relocate lignin, thereby increasing the accessibility of the remaining cellulose. The addition of an acid catalyst prior to steam treatment is commonly used to increase hydrolysis of hemicelluloses. However, residual lignin in the treated biomass is known to non-specifically bind to hydrolytic enzymes, thereby reducing the efficiency of cellulose hydrolysis. Steam treatment of biomass, particularly acid treated biomass, also releases inhibitory compounds, including phenolic compounds that can both inhibit enzymatic hydrolysis of cellulose to glucose and its subsequent fermentation to produce alcohol.
Laccase treatment of steam treated biomass has been investigated for its ability to improve the down-stream processing of biomass to produce fermentable sugars and alcohol. Laccase (benzenediol: dioxygen oxidoreductases; EC 1.10.3.2) is a blue copper containing enzyme, which catalyzes the removal of an electron and a proton from phenolic hydroxyl or aromatic amino groups to form free phenoxy radicals and amino radicals, respectively. During this reaction, one molecule of atmospheric oxygen is reduced to two molecules of water. Laccase, acting via a mediator, is also able to oxidize non-phenolic lignin units (C4-esterified) to radicals.
Treatment of steam-pretreated softwood with a laccase from Trametes hirsuta is reported to improve its enzymatic hydrolysis, however the efficacy of laccase treatment was partly dependent on the presence of the mediator, N-hydroxy-N-phenylacetamide (Palonen and Viikari, 2004). Two laccase enzymes, isolated from Cerrena unicolor and Trametes hirsuta, are reported to enhance cellulose hydrolysis of steam-treated spuce wood, but to inhibit cellulose hydrolysis of steam-treated giant reed (Moilanen et al., 2011). Treatment of steam-exploded wheat straw with laccase is reported to cause lignin polymerization, which may contribute to detoxification of the products released by stream treatment (Jurado et al., 2009). However, these investigators report that treatment with Coriolopsis rigida laccase when performed prior to enzymatic hydrolysis of cellulose actually reduced the recovery of glucose, which was attributed to the release of phenolic compounds by laccases that inhibit cellulases. The investigators comment that contradictory results are reported with respect to laccase treatment, and they conclude that detoxification methods must be studied independently for each pretreated material.
There exists a large industrial interest in identifying new sources of enzymes that can efficiently degrade lignin, and enhance glucose release during lignocellulose degradation (hydrolysis). A search for phylogenetically-related laccases reveals that a large number of different laccases are produced by white-rot fungi (Basiodiomycota). Ganoderma lucidum (lingzhi) is one of the many members of the white-rot fungi. It is well known as a medicinal mushroom in traditional Chinese medicine and is commonly used for pharmaceutical purposes and in health foods.
A laccase gene GLlac1, encoding a GLlac1 laccase, has been cloned from Ganoderma lucidum (strain 7071-9 monokaryon) and expressed in Pichia pastoris. The expressed laccase is reported to confer anti-oxidative protection from protein degradation, with potential biomedical applications (Joo et al., 2008). GLlac1 laccase, when expressed from a synthetic gene GILCC1 in Pichia pastoris, has a Km of 0.995 mM, a pH optimum of 2.6 and is reported to be useful for the removal of color from reactive textile dye effluent (Sun et al., 2012). A laccase, has been isolated from Ganoderma lucidum fruiting bodies, having a molecular mass of 75 kDa, and the N-terminal sequence, GQNGDAVP, and is reported to be capable of inhibiting HIV-1 RT (Wang and Ng, 2006). A laccase, isolated from Ganoderma lucidum and reported to be useful in paper-making, is disclosed in CN1657611A.
Efficient use of lignocellulose biomass, in particular sugarcane bagasse, as a renewable source of bioethanol is dependent on the provision of individual lignocellulose modifying and degrading enzymes that in combination with optimal pre-treatment steps can maximize fermentable sugar yields. Chandler et al 2007 report that detoxification of sugarcane bagasse hydrolysate using a laccase isolated from Cyathus stercoreus NCIM 3501 can improve ethanol fermentation by Candida shehatae. There remains a need however, to provide enzymes that can enhance the yield of fermentable sugar yields from biomass, in particular steam exploded sugar cane bagasse, such that the use of this biomass can become economically viable.